Strategies for Proteomics

Bottom-up proteomics. This strategy relies on peptide-level information, such as mass or sequence, to identify a protein. Cnrrently, this strategy dominates proteomics research. In this approach, the protein is digested, typically with trypsin, and the digest is analyzed with an appropriate mass spectrometry system to obtain the peptide masses and sequences. The database search with mass spectrometry data characterizes the protein. More details of the mass spectrometry platforms used in this approach are provided in the following sections. 

Top-down proteomics. This strategy deals with intact protein molecules no proteolytic cleavage is performed [41], It involves the accurate molecular mass measurement of the intact protein nsing high-resolution mass spectrometry within 2 Da, followed by a molecular mass database search. The identity of 

The field of proteome analysis is challenging because of the complexity of any given proteome, the broad dynamic range of protein abundance, the existence of mnltiple protein forms dne to PTMs, proteolysis and splice variants, and the dif-flcnlty of quantification of absolute protein content. Some of these challenges can be met by the use of complementary approaches to proteomics. In general, four basic steps are involved in proteome analysis (1) sample preparation, (2) separation and purification, (3) mass spectrometry analysis, and (4) database search. 

Separation and Purification A high degree of purification of the homogenized subcellular fractionations into individual proteins (rarely achieved even by two-dimensional-PAGE for all proteins) or a simple mixture is an essential requirement for unambiguous protein identification. This aspect was also discussed in Section 8.3. The use of several orthogonal techniques (e.g., 2-DE or LC) becomes essential to reduce the complexity of the sample and improve chances of detection of low-abundance proteins. 

Peptide-mass fingerprinting. In this approach, also known as peptide-mass mapping, the protein is first subjected to enzymatic digestion to generate a set of peptides that are unique to this protein (Section 8.4) [20,42-47]. The molecular mass of each fragment is determined accurately (within 0.5 Da) using MALDI-MS or ESI-MS. Correlation of these masses with the theoretical peptide 

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Wang, H., Hanash, S. (2005). Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry. Mass Spectrom. Rev. 24, 413 126. 

Leitner, A., and Lindner, W. (2004) Current chemical tagging strategies for proteome analysis by mass spectrometry./. Chrom. B813, 1-26. 

To address this problem, recently a new strategy for proteome analysis has emerged. This technology, named Combinatorial Proteomic, uses antibody libraries as probes to profile the expression and function of protein families in complex proteomes. The use of antibodies allows the detection of iper- and ipo-expressed proteins, even if they are at pico-quantity level, overcoming one of the proteomic limitations of difficulty in detecting low abundance proteins [46, 47],... 

Liu, P., Regnier, F. E. (2002). An isotope coding strategy for proteomics involving both amine and carboxyl group labeling. J. Proteome Res. 1, 4434-50. 

Figure 18 Glycoprotein-enrichment strategies for proteomics. (a) Lectin affinity chromatography (b) chemoenzymatic modification of 0-GlcNAc-modified proteins with biotinylated probes for proteomic analysis after avidin chromatography.

Wait, R., Miller, I., Eberini, I., Cairoli, F., Veronesi, C., Battocchio, M., Gemeiner, M., and Gianazza, E., 2002, Strategies for proteomics with incompletely characterized genomes the proteome of Bos taurus semm. Electrophoresis 23 3418-3427. 

Proteomics. Figure 2 A strategy for mass spectrometry (MS)-based identification of proteins. 

Ji, J. Chakraborty A. Geng, M. Zhang, X. Amini, A. Bina, M. Regnier, F. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. B Biomed. Sci. Appl. 2000, 745,197-210. 

The present chapter does not consider analysis of extracted protein biomarkers but rather focuses on strategies for rapid chemotaxonomic analysis of intact microorganisms with automated sample manipulation. Rapid means less than 5 minutes. Advantages of the application of bioinformatics and proteomics strategies for rapid identification of microorganisms include the following ... 

Kislinger, T., Rahman, K., Radulovic, D., Cox, B., Rossant, J., Emili, A. (2003). PRISM, a generic large scale proteomic investigation strategy for mammals. Mol. Cell. Proteomics 2, 96-106. 

Bjorhall, K., Miliotis, T., Davidsson, P. (2005). Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples. Proteomics 5, 307-317. 

Sadaghiani AM, Verhelst SHL, Bogyo M (2007) Tagging and detection strategies for activity-based proteomics. Curr Opin Chem Biol 11 20-28... 

Celis JE et al. Proteomics and immuno-histochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium a novel strategy for identifying metaplastic lesions. Cancer Res 1999 59 3003-3009. 

Speers, A.E., and Cravatt, B.F. (2004a) Chemical strategies for activity-based proteomics. Chem. Bio. Chem. 5, 41 17. 

Zappacosta, F., and Annan, R.S. (2004) N-terminal isotope tagging strategy for quantitative proteomics Results-driven analysis of protein abundance changes. Anal. Chem. 76, 6618-6627. 

Zhang, H., Yan, W., and Aebersold, R. (2004) Chemical probes and tandem mass spectrometry A strategy for the quantitative analysis of proteomes and subproteomes. Curr. Opin. Chem. Biol. 8, 66-75. 

General Strategy for Mass Spectrometry-Based Proteomics... 

F. tularensis [18] and Shigella flexneri [19]. Once immunoreactive proteins are identified, these proteins are evaluated for their potential as vaccine candidates using a multifaceted algorithm [11]. An overall strategy in proteomics-based vaccine discovery is presented in Fig. 12.2. 

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